This invention pertains to a method for preparing packed capillary columns. More particularly, the invention pertains to a multi-step method of packing capillary tubes to form packed capillary columns wherein a pre-pack column packed with particulate materials is first prepared by the slurry packing method, and then the packing is transferred from the pre-pack column to a second capillary tube to form a final packed capillary column.
Capillary columns are capillary channels which have been packed with a packing material. Suitable channels may be fabricated from hollow tubing of appropriate diameter or formed in planer substrates through a variety of processes.
There are a variety of methods currently in use for packing capillary channels to form packed capillary columns, such as those columns used in the fields of chromatography and electrospray ionization mass spectrometry (ESI-MS).
One method for packing capillary tubes is known as the xe2x80x9cDry Packing Methodxe2x80x9d. In accordance with this method, dry packing material, such as glass, silica, polymeric powder or metallic powder, is forced into one end of capillary tube. In a particularly advantages aspect of this method, the particulate materials are rapidly vibrated as they are loaded into the tube through a funnel.
Narrow-bore columns, which are being found useful for an expanding variety of technological applications, have inside diameters which are generally  less than 300 xcexcm, and typically constructed of steel, polymer or fused-silica. Especially narrow columns will be required for the so-called xe2x80x9clab on a chip devicesxe2x80x9d which are now in their early stages of development, in which capillary channels are fabricated in planar substrates, such as glass or silicon wafers. The xe2x80x9cDry Packing Methodxe2x80x9d is unsatisfactory for loading such columns, because the small diameters involved do not allow for the free flow of dry powdered material.
A second method for packing capillary tubes is known as the xe2x80x9cSlurry Packing Methodxe2x80x9d. In accordance with this method, a slurry, i.e., a liquid comprising suspended particles of packing material, is forced under pressure into the proximal end of the tube, and pumped until the slurry reaches a frit at the distal end of the tube. The frit serves to xe2x80x9cfilterxe2x80x9d the particulate packing material from the liquid, also known as the xe2x80x9cmobilexe2x80x9d phase. The mobile phase thus passes through the frit and out of the tube, while the solid packing particles remain behind the frit. As the tube begins to thus become packed, the back-pressure on the system increases due to viscous flow. The packing rate, and the flow rate of the mobile phase through the tube, thus decreases as packing progresses and the amount of packing built-up behind the frit increases. In order to compensate for the increased back-pressure, and maintain a constant flow rate, the pressure of the slurry entering the tube has to be increased.
Slurry packing normally requires the use of high pressures ( greater than 1000 psi) in order to generate a high flow rate of mobile phase and resultant high xe2x80x9cimpact velocityxe2x80x9d of the incoming particles. This high velocity forces the incoming particles into intimate contact with the bed. In this way, a tightly packed bed is formed. A tightly packed bed is important for good, reproducible chromatographic performance. This is especially important for column-to-column reproducibility. Slurry packing can be utilized to form columns in capillary channels that are frbricated in tubular or planar substrates.
The xe2x80x9cSlurry Packing Methodxe2x80x9d, while useful, generally requires the use of expensive instrumentation capable of generating and withstanding high operating pressures. This becomes much more the case as the trend towards columns having smaller and smaller inside diameters continues.
In yet a third method, the channel is filled with a monomer solution or a gel, and then the monomer is caused to polymerize inside the tube, to form a continuous porous bed through which gas or liquid may then flow. No solid material is initially introduced into the channel, and this method is based on a change in the state of the initial material charged into the channel from a liquid or gel into a porous solid.
Other known methods involve electroosmotic packing, centrifugal packing and evaporative packing.
None of the foregoing methods offer the economy and ease of use of the method we have now discovered.
We have now discovered a method for column packing based on the traditional slurry method, which is compatible with low packing pressures (xe2x89xa61000 psi) and narrow bore columns ( less than 300 xcexcm inside diameter). This method utilizes a multi-step approach that is analogous to an xe2x80x9cannealingxe2x80x9d or condensation process. In xe2x80x9cannealingxe2x80x9d, residual stress or defects in a system are removed through the application of energy.
In accordance with the method, a pre-pack channel is packed with a packing material by the slurry method to form a pre-pack column. The frit is then removed from the pre-pack column, and the pre-pack column is then joined with the final channel to be packed. A fluid, such as that used as the mobile phase for the slurry packing of the pre-pack column, is then forced through the pre-pack column and into the final channel, whereby the packed material in the pre-pack column flows into the final channel to form a packed bed in the final channel, thereby forming a final packed column.
In accordance with the method of the present invention, a channel having a proximal end and a distal end, with a porous frit at the distal end, is first pre-packed by forcing a slurry of packing material packing into the proximal end of the channel at a slurry pressure in the range of from about 100 to about 1,000 psi. A loosely-packed bed is thereby formed in the channel, to form a pre-pack column. This loosely-packed bed, however, typically has a plurality of packing defects. Such packing defects are characterized as undesirably large void spaces within the bed. The presence of such large voids in a packed bed would cause the bed to perform poorly in chromatography service.
The frit is then removed from the distal end of the pre-pack column and the pre-pack column, or a portion thereof, is joined to the proximal end of a second channel, void of any mobile phase, having a proximal end and a distal end and having a porous frit at the distal end. The distal end of the pre-pack column is preferably secured to the proximal end of the second channel by a liquid-tight, zero dead volume, seal using a xe2x80x9cunionxe2x80x9d; although any of the other types of devices known in the art for securing one channel to another or any other method of joining one tube to another may also be used for this purpose.
The second channel initially is empty of any liquid, although the presence of a gas, such as air, nitrogen, helium, argon or the like may be desirable. It is especially desirable to have Helium present in the second channel, as Helium is highly compressible and leads to faster travel of the slurry through the channel.
A liquid, such as the mobile phase liquid used in the slurry, is then forced through the proximal end of the pre-pack column, to force the bed of packing material out of the pre-pack column and into the second channel. As the bed flows into the second tube and flows from the proximal end of the second tube to the distal end, kinetic energy from the flowing liquid phase induces transient contact of the particles making up the bed with each other, and induces a uniform distribution of the particles within the bed. As the bed of packing material reaches the frit at the distal end of the second channel, a re-packing of the bed takes place. The re-packing takes place much more quickly than did the pre-packing, because the packing velocities of the individual particles are more uniform; that is to say, that the velocity of each individual particle will be close to the velocity of each of the other particles, so that there will be a uniform velocity profile of the particles as they move through the channel towards the frit. This has the effect of reducing the spaces between the individual particles, so that when packing takes place the amount and size of gaps between the packed particles is reduced, for a more uniform packing having less defects than has heretofore been achievable.
In a further embodiment of the invention, several pre-pack columns are stacked in succession prior to packing the xe2x80x9csecondxe2x80x9d channel to create the packed column. This embodiment enables the preparation of longer packed columns, i.e., longer packed lengths; as well as columns having sections of different kinds of packing materials in succession.
Optionally, if further improvement in the packing is desired, the packed xe2x80x9csecondxe2x80x9d column can then be used as a new xe2x80x9cpre-packxe2x80x9d column, with a new empty xe2x80x9csecondxe2x80x9d channel, and the process can be repeated. This can be repeated as many times as desired, until a point is reached where further processing yields diminishing degrees of improvement in the packing.
There are a variety of techniques that can be used to force the mobile phase through the distal end of the pre-pack column, and the invention is not limited to any particular method. By way of example, the slurry can be forced into the pre-pack channel by applying a gas pressure, such as air or nitrogen pressure, to the slurry itself. A vacuum can also be applied to the distal end of the pre-pack tube to xe2x80x9cdrawxe2x80x9d the slurry in, or both a pressure on the slurry at the proximal end of the channel and a vacuum on the distal end of the channel can be used.
The packing materials used may be particles of a variety of shapes, such as spherical, hemispherical, xe2x80x9cirregularxe2x80x9d spheres, rods with aspect ratios of  less than 5:1, fractured xe2x80x9cchipsxe2x80x9d (i.e., shapes associated with finely ground materials), precipitated crystallites (tiny cubes, prisms, dodecahedral, etc.) or powders. Spherical or nearly spherical shapes are preferred, however, since such shapes allow for the most uniform and dense packing. The packing materials may be solid, hollow or porous such as, for example, solid, hollow or porous spheres.
Preferred packing materials are ceramic, metallic or polymeric. The ceramic materials which can be used include, for example, soda-lime glass, borosilicate glass, porous silica (silica gel) and non-porous silica. The metals which can be used include, for example, colloidal gold, colloidal silver, nickel and stainless steel. The polymeric materials which can be used include, for example, fluoropolymers, such as polyvinylidene fluoride (PVDF), fluorinated ethylene propylene (FEP); styrenics, such as polystyrene (PS) and polystyrene/divinylbenzene copolymer (PS/DVB); polyolefins such as high density linear polyethylene (HDPE), low-density linear polyethylene (LDPE) and polypropylene; polyketones, such as polyetheretherketone (PEEK); acrylics, such as polymethylmethacrylate (PMMA) and vinyls, such as divinylbenzene (DVB). Particularly preferred materials are borosilicate glass, silica (both porous silica and non-porous silica) and PS/DVB copolymer.
The particles which are used should have dimensions, i.e., diameters in the case of spheres, which are smaller than the smallest internal dimension of the channel to be used, if the channel has an internal shape other than round; or smaller than the internal diameter of the channel, if the channel to be used has a round internal shape; and should have maximum dimensions, or diameters if spherical, of about xc2xd the smallest internal dimension or diameter of the channels used. In general, the largest dimensions of non-spherical particles, or the diameters of the spherical particles used, range from about 0.1 xcexcm to about 1 mm, although a range of 0.25 xcexcm to about 250 xcexcm is preferred; a range of 0.5 to 30 xcexcm being particularly preferred, a range of 1 to 5 xcexcm being especially preferred.
There are many liquids known to the art which can be used as a mobile phase to form the slurry. Preferred liquids are methanol, ethanol, isopropanol, methylene chloride, acetone, acetonitrile, tetrahydrdrofuran (THF) and water; although almost any liquid can be used, as long as it is not harmful to the packing material or tube. The liquid selected should thus be one that will not dissolve, swell or otherwise harm the packing material selected, although it should xe2x80x9cwetxe2x80x9d the surface of the packing material.
The channels which are used are those known to the art, and can, for example, be those which are generally classified as ceramics, such as borosilicate glass, fused-silica, polyimide coated fused-silica and aluminum coated fused-silica; metallic, such as stainless steel, glass lined stainless steel or silica lined stainless steel; or they can be of polymeric materials. The polymeric material which can be used include fluoropolymers, such as ethylene tetrafluoroethylene (ETFE), fluorinated ethylene propylene (FEP) and polytetrafluoroethylene(PTFE); polyolefins, such as high density linear polyethylene (HDPE), low-density linear polyethylene (LDPE) and polypropylene; polyketones, such as polyetheretherketone (PEEK) and silica-lined PEEK; acrylics, such as polymethylmethacrylate (PMMA), polyamides, such as nylon 6, nylon 11 and nylon 12; and polyimide. Preferred tubes in accordance with the invention are those of polyamide-coated fused silica, stainless steel, PEEK and HDPE, although polyimide-coated fused silica is especially preferred.
The internal or external shapes of the channels used in the practice of this invention can take on a variety of regular geometric shapes, such as round, oval, square, rectangular, polygonal, such as pentagonal, hexagonal, and the like; or can take on irregular shapes. The term xe2x80x9cinternal shapexe2x80x9d of the channels, as used herein, has the same sense of meaning as the xe2x80x9cborexe2x80x9d of a tube. Particularly preferred are those channels having a round internal shape or bore.
The channels used in the practice of the invention, having round internal shapes or bores, have inside diameters in the range of from about 1 xcexcm to about 5 mm, preferably 10 xcexcm to 2 mm, and particularly preferably 500 xcexcm to 1 mm, especially tubes having inside diameters of about 75 xcexcm to about 300 xcexcm. Where tubes having internal shapes other than round are used, their internal cross-sectional areas should be in the same range as that of a tube having a round internal shape with a diameter in the range of from about 10 xcexcm to about 2 mm, preferably that of a tube having a round internal shape with a diameter 50 to 250 xcexcm and particularly preferably that of a tube having a round internal shape with a diameter 75 to 300 xcexcm
The tubes or channels can be of uniform internal dimensions or diameter over their entire length, such as those typically used for chromatography columns, or they can be tapered at one end, so that the internal diameter tapers to a narrow tip or needle, such as those columns used for electrospray ionization mass spectrometry (ESI-MS). The columns having tapered ends are also referred to in the art as needles.
The tubes or channels used for the pre-pack and second tubes can be of the same type as each other, or different. Thus, the diameter, length, cross-section, and materials of construction of one can each be independently different than that the other.
The length of the pre-pack and second columns to be used will vary with the contemplated application, as well as the amount of additional packing, if any, which is to be used in combination with the packing of the present invention. That is to say, the packing of the present invention can be used alone, or in combination with other packings which can be added to the column before or after the present packing. Packed columns with lengths of 19 meters or more are known (U.S. Pat. No. 4,793,920), and such columns can be used in the practice of this invention, for which the length of the column used is not limited.
The slurry can be prepared by conventional methods, known to those skilled in the art. One such method is simple mixing, wherein a liquid is introduced into a vessel, such as a vial, beaker or a flask, together with the packing material, and the contents are then stirred.